LncRNA HOTAIR promotes the proliferation and invasion/metastasis of breast cancer cells by targeting the miR-130a-3p/Suv39H1 axis

Long noncoding RNAs (lncRNAs) are a group of transcripts, more than 200 bp in size and regulate cell proliferation, differentiation and apoptosis. LncRNA HOX Transcript Antisense Intergenic RNA (HOTAIR) promotes tumor progression and increases cancer susceptibility by regulating microRNA expression and function. HOTAIR regulates miR-130a-3p expression in hepatocellular carcinoma cells. Bioinformatics analysis revealed that Suv39H1 contained a putative binding site for miR-130a-3p. We speculate that LncRNA HOTAIR promotes the proliferation and invasion/metastasis of breast cancer (BC) cells by targeting the miR-130a-3p/Suv39H1 axis. High HOTAIR expression facilitated BC cell growth and metastasis. HOTAIR functioned as a ceRNA by sponging miR-130a-3p and subsequently promoted Suv39H1-mediated AKT/mTOR signaling. Suv39H1 restoration abolished the effects of HOTAIR knockdown on BC cell growth and metastasis. HOTAIR facilitated the Suv39H1-mediated AKT/mTOR pathway by acting as a molecular sponge of miR-130a-3p.Our results provide a better understanding of the interactions of HOTAIR and miR-103a-3p/Suv39H1 in BC and a potential prognostic biomarker and therapeutic target for BC.


Introduction
Breast cancer (BC) is the most common cancer among women and ranks second among the main causes of tumor-related deaths in women [1]The prognosis of patients with BC remains unsatisfactory worldwide despite advances in diagnosis and combined treatments [2].Long noncoding RNAs (lncRNAs), a group of transcripts, more than 200 bp in size, are involved in molecular diagnosis and therapy [3,4]. LncRNA HOX Transcript Antisense Intergenic RNA (HOTAIR), as an oncogenic trans-acting lncRNA, is elevated in multiple cancers [5] and plays a regulatory role in the growth, apoptosis, migration and invasion of carcinoma [6,7].However, the underlying molecular mechanism of HOTAIR in BC remains not yet fully elucidated.HOTAIR regulates miR-130a-3p expression in hepatocellular carcinoma cells [8]. Bioinformatics analysis from TargetScan Human Release 7.2 (http://www. targetscan.org/vert_72/) showed that Suv39H1 contained a putative binding site for miR-130a-3p. In this study, we speculate that LncRNA HOTAIR promotes the proliferation and invasion/metastasis of BC cells by targeting the miR-130a-3p/Suv39H1 axis.

1 patients and specimens
Twenty pairs of human BC tissue specimens and corresponding paracarcinoma normal tissue specimens were collected from patients admitted to Jiangxi Cancer Hospital of Nanchang University (Nanchang, China) from January 2018 to May 2021. No patient received radiotherapy and chemotherapy before the operation. Written informed consents were obtained from all patients. This research was approved by the Ethical Committee on Scientific Research of Jiangxi Cancer Hospital of Nanchang University.

Cell culture
BC cell lines SKBR3,MCF-7 and MDA-MB-231 and human normal mammary epithelial cell line MCF10A were obtained respectively from the Cell Bank of Type Culture Collection of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. All in vitro cell lines were maintained in Roswell Park Memorial Institute-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin, Sigma, St-Louis, MO, USA) in a humidified tissue culture chamber at 37 • C.Cells in the logarithmic growth period were used for subsequent experiments.

Transfection
The lentivector-mediated HOTAIR (overexpression/HOTAIR and silencing sequences/si-HOTAIR) and negative control (Vector and NCsi) were designed by Shanghai Gene Pharma Company (Shanghai, China). BC cells were transduced with lentiviruses in the presence of polybrene (Sigma). The sequences of HOTAIR and Suv39H1 cDNA were PCR amplified using a PCR Master Mix reagent kit (Thermo Fisher Scientific, USA) and then cloned into the pcDNA3.1 plasmid (Invitrogen). MiR-130a-3p mimics, miR-130a-3p inhibitors, and corresponding negative controls were purchased from Shanghai GenePharma Co., Ltd. (China). BC cells were seeded into 96-well plates (4 × 10 4 cells/well) in accordance with the manufacturer's protocol. Cell transfection was performed using Lipofectamine® 2000 (Invitrogen), and the cells were incubated at 37 • C in 5% CO 2 .

Western blot analysis
Tumor tissues or cells were lysed with Sigma-Aldrich RIPA buffer. Total protein was quantified by using a BCA protein assay kit (Abcam). Equal amounts of protein samples were separated by 10% SDS-PAGE gel and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were subsequently incubated with primary antibodies overnight at 4 • C and then incubated with secondary antibodies for 1 h at room temperature.Then, the membranes were visualized with electrochemiluminescence reagents and imaged. The results were analyzed by Image J version 1.8.0 (National Institutes of Health).

Cell proliferation assay
Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8) assay in accordance with the manufacturer's instructions. CCK-8 reagent was added to the culture medium, and the transfected BC cells in a 96well plate were incubated at 37 • C for 3 h. Absorbance at 450 nm was recorded for each experiment and normalized to control by using a spectraMax Gemini-XPS microplate reader (Molecular Devices, USA). The optical density ratio of each well indicated cell vitality. The entire experiment was repeated three times.

Colony formation assay
BC cells in the logarithmic growth phase were seeded in six-well plates at a density of 1200 cells/well and then cultured a humidified incubator at 37 • C. After 2 weeks incubation, the cells were washed and fixed in 4% fixative solution (Solarbio, China) for 15 min. The number of visible cell colonies was counted to evaluate cell proliferation.

Transwell invasion and migration assays
Cell migration and invasion were assessed using trans-well filters (8 μm pores; Corning, Corning, NY, USA) coated without or with Matrigel (BD Biosciences, NJ, USA) in accordance with the manufacturer's instructions. After transfection, the BC cells were suspended in serum-free medium in the upper chambers. Medium supplemented with 10% fetal bovine serum was added into the lower chamber. After incubation at 37 • C for 12-24 h, the cells on the upper surface of the filters were removed with a cotton swab. The cells underside of the membrane were fixed and dyed on the lower surface of the membrane with hematoxylin and eosin. The membrane was photographed using a colored digital camera connected to a microscope. For migration assay, similar steps were conducted but with the absence of Matrigel coating.

Tumor xenograft model
After the cells were stably transfected with si-HOTAIR or NC-si, SKBR3 cells were injected subcutaneously into the mammary fat pad of 6-week-old BALB/c nu/nu female mice (1 × 10 6 cells per injection, n = 5 per group). The mice were used according to the guidelines of the Chinese Medical Research Commission and the experiments were approved by the Ethical Committee on Scientific Research of Jiangxi Cancer Hospital of Nanchang University (No. 20180314). Tumor growth was monitored by caliper measurements. The length and width of the tumors were measured. Tumor volume was calculated as 0.5 × length × width 2 . The mice were sacrificed 32 days after implantation.

Pull-down assay with biotinylated miR-130a-3p
BC cells were transfected with biotinylated wild-type (wt) miR-130a-3p, mutant (mt) miR-130a-3p, or NC (Ribo Biotech). The biotinylated RNA complex was pulled down by incubating the cell lysates with streptavidin-coated magnetic beads at 4 • C on the rotator overnight in accordance with the manufacturer's instructions. Then, the beads were washed three times with ice-cold lysis buffer and once with high-salt buffer. The bound RNAs were purified using TRIzol for qRT-PCR analysis.

Luciferase reporter assays
The 3 ′ -UTR sequences of Suv39H1 and HOTAIR were cloned and inserted into the psiCHECK2 vector encoding dual luciferase. The potential miR-130a-3p binding sequences were mutated using the Quik-Change® Site-Directed Mutagenesis Kit (Santa Clara). The wt (mt) 3 ′ -UTR of the Suv39H1 vector or wt (mt) HOTAIR vector and miR-130a-3p or control mimics were co-transfected into SKBR3 cells. Luciferase activity was determined using GeneCopoeia's Luc-Pair™ Duo-Luciferase HS Assay Kit.

RNA immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) assay was performed using the Thermo Fisher RIP kit (Thermo Fisher Scientific, MA, USA) in accordance with the manufacturer's protocol. SKBR3 cells were lysed using the complete RIP lysis buffer, and magnetic beads were pre-incubated with a human anti-AGO2 antibody or with a negative control normal mouse anti-IgG. RNA was then purified and detected through qRT-PCR.

Statistical analysis
Data are presented as mean ± SD from three independent experiments and analyzed using GraphPad Prism 6.0 Software. Statistical difference was measured using Student's t-test or one-way ANOVA. Overall survival curves were protracted using the Kaplan-Meier method and estimated by the log-rank test. Statistical significance was considered at p < 0.05.

Levels of lncRNA HOTAIR and Suv39H1 are increased in BC
The Cancer Genome Atlas (TCGA,http://ualcan.path.uab.edu/anal ysis.html) data from UALCAN were employed to investigate differentially expressed LncRNA HOTAIR and Suv39H1 in breast carcinoma. Bioinformatics analysis revealed that the expressions of HOTAIR and Suv39H1 were higher in tumor tissues than those in noncancerous tissues (Fig. 1A). The expressions of HOTAIR and Suv39H1 in BC cell lines and 20 pairs of BC and matched tumor-adjacent normal tissues were measured using qRT-PCR to verify the clinical significance of LncRNA HOTAIR and Suv39H1 in BC. HOTAIR and Suv39H1 markedly increased in BC (Fig. 1B-C). These results indicate that HOTAIR and Suv39H1 are highly expressed in patients with BC.

HOTAIR/Suv39H1 facilitates BC cell proliferation, invasion, and metastasis
The lentivector-mediated HOTAIR was used in BC cells to determine the role of HOTAIR in BC. The levels of HOTAIR were downregulated in SKBR3 cells and upregulated in MCF-7 cells, respectively (p < 0.05, Fig. 3A). CCK-8 and colony formation assays showed that HOTAIR depletion significantly inhibited the proliferation of SKBR3 cells, whereas HOTAIR overexpression promoted the proliferation of MCF-7 cells (p < 0.05, Fig. 3B-C). Transwell invasion and migration assays indicated that silencing HOTAIR noticeably suppressed whereas HOTAIR overexpression increased the migration and invasion of BC cells (p < 0.05, Fig. 3D). The results indicate that HOTAIR facilitates the growth and metastasis of BC cells in vitro.

Silencing HOTAIR inhibits cancer proliferation/metastasis and decreases Suv39H1 expression in vivo
SKBR3 cells with or without HOTAIR silencing were injected into nude mice subcutaneously to examine the effects of HOTAIR in vivo. Tumor volume and growth were used to observe the tumor growth status. The tumor volume and weight in the HOTAIR silencing group were apparently lower than those in the NC si group (p < 0.05, Fig. 4A). Western blot analysis indicated that Suv39H1 levels significantly decreased in tumor tissues of the HOTAIR silencing group (p < 0.05, Fig. 4B-C). These findings suggest that HOTAIR silencing suppresses the tumorigenesis, growth, and metastasis of BC cells in vivo.

HOTAIR acts as a competing endogenous RNA (ceRNA) by sponging miR-130a-3p
LncRNAs usually function as ceRNAs and serve as miRNA sponges to inhibit miRNAs [9]. HOTAIR silencing significantly upregulated miR-130a-3p expression, whereas HOTAIR overexpression decreased miR-130a-3p level in BC cells (p < 0.05, Fig. 5A-B). Moreover, luciferase reporter assay indicated that the forced expression of miR-130a-3p strikingly reduced the fluorescence intensity of the vector containing wt HOTAIR rather than the vector containing mt HOTAIR (p < 0.05, Fig. 5C). Furthermore, HOTAIR was pulled down by biotinylated miR-130a-3p, whereas mutagenesis of the binding sites for HOTAIR in miR-130a-3p abolished this interaction (p < 0.05, Fig. 5D). AGO2, as a core element of the RNA-induced silencing complex, acts as an important regulator of miRNA function [10]. The levels of HOTAIR and miR-130a-3p were highly enriched in BC cells, as suggested by the RIP assay using an AGO2 primary antibody (p < 0.05, Fig. 5E). Moreover, AGO2 silencing enhanced HOTAIR expression and reduced miR-130a-3p level in BC cells (p < 0.05, Fig. 5F). These results suggest that HOTAIR acts as a miR-130a-3p sponge in BC cells.

Suv39H1 is a novel target of miR-130a-3p in BC cells
TargetScan was used to predict the miR-130a-3p binding sites in 3 ′ -UTR of Suv39H1 mRNA, and only one potential complementary sequence was found for miR-130a-3p at nt 437-443 (Fig. 5G). Then, the forced expression of miR-130a-3p decreased while miR-130a-3p silencing elevated Suv39H1 mRNA and protein expression in BC cells (p < 0.05, Fig. 5H and I). Luciferase reporter assay indicated that miR-130a-3p overexpression apparently reduced the fluorescence intensity of the vector containing the wt 3 ′ -UTR of Suv39H1 rather than the mt 3 ′ UTR of Suv39H1 (p < 0.05, Fig. 5J). Increased level of HOTAIR enhanced the level of Suv39H1, which was subsequently reduced by the increased level of miR-130a-3p in SKBR3 cells. Forced upregulation of miR-130a-3p remarkably reduced the level of Suv39H1, whereas this effect was reversed by elevated HOTAIR (p < 0.05, Fig. 5K). These results indicate that HOTAIR induces Suv39H1 expression by sponging miR-130a-3p in BC cells.

Discussion
Elevated expression of HOTAIR/Suv39H1 was confirmed in our BC tissues and cell lines, which were bioinformatically analyzed in patients with BC (vs. Normal breast tissues). HOTAIR level increased in the patients with N3 (vs N0-1). Suv39H1 expression was enhanced in the patients with Triple Negative and HER2-enriched (vs. Luminal)/Stage 2-3 (vs. Stage1). HOTAIR promotes the proliferation, invasion, and metastasis of human cancer cells [12]. LncRNA HOTAIR indicates its progression and predicates metastasis risk in breast carcinoma [13]. HOTAIR is related to the poor overall survival of patients with ovarian cancer [14]. Collectively, these studies indicate that HOTAIR level increases in human cancer and is related to metastasis and poor prognosis.
Suv39H1 promotes tumor growth in human carcinoma, and pharmacological suppression of Suv39H1 is an effective approach to inhibit human carcinoma [15,16]. Suv39H1 may regulate the proliferation and metastasis of epithelial ovarian cancer cells [17]. Suv39H1 reduces cytotoxic T lymphocyte effector gene expression and causes colon cancer immune escape [18]. Suv39H1 knockdown represses cell proliferation; and MCF-7 cells that were transfected with two different si-HOTAIR and HOTAIR vector respectively, were subjected to qRT-PCR for miR-130a-3p expression. HOTAIR down-regulated the expression of miR-130a-3p in BC cells. (C) After co-transfection with miR-130a-3p mimics and vector containing wild type (wt) or mutated (mt) HOTAIR, levels of luciferase activity were measured. MiR-130a-3p mimics transfection inhibited the luciferase activity of the WT but not the MUT HOTAIR reporter in BC cells.(D) HOTAIR was pulled down by biotin labelled wt or mt miR-130a-3p. HOTAIR was highly enriched in the sample pulled down by biotinylated wt miR-130a-3p rather than mt miR-130a-3p. (E). Anti-AGO2 RIP was performed in BC cells, followed by RT-PCR to validate the level of LncRNAHOTAIR or miR-130a-3p associated with AGO2.(F)SKBR3 cells that were transfected with AGO2 siRNA or NC siRNA were subjected to qRT-PCR for HOTAIR and miR-130a-3p expression.(G-K)Suv39H1 is a direct target of miR-130a-3p in BC cells.(G)Binding site of miR-130a-3p and Suv39H1 3 ′ -UTR according to online bioinformatics analysis.(H-I) BC cells were transfected with miR-130a-3p mimics and miR-130a-3p inhibitors, respectively. Suv39H1 expression were detected via qRT-PCR and immunoblotting. (J) Forced expression of miR-130a-3p decreased the fluorescence intensity of the vector containing wild type (wt) 3 ′ UTR of Suv39H1, but did not affect the fluorescence intensity of vector containing mutated (mt) 3 ′ UTR of Suv39H1⋅(K)Increased expression of HOTAIR enhanced the level of Suv39H1, which was reduced by miR-130a-3p overexpression in BC cells. MiR-130a-3p restoration decreased the expression of Suv39H1, and this effect was reversed by increased HOTAIR in BC cells. (*p < 0.05 by Student's t-test, #p < 0.05 by ANOVA. n = three independent repeats.). thus, Suv39H1 might be a promising target for cancer therapy [19]. Subcutaneous tumor formation model in nude mice demonstrated that HOTAIR knockdown inhibited the tumor growth and metastasis of BC in vivo. MiR-130a-3p is decreased in esophageal squamous cell carcinoma and suppresses the migration and invasion of esophageal squamous cancer cells in vitro and in vivo [20]. In the present study, HOTAIR inversely regulated the level of miR-130a-3p in BC cells ( Fig. 5A and B). Further experiments identified that HOTAIR acted as a ceRNA by sponging miR-130a-3p. MiR-130a-3p downregulated Suv39H1 in BC cells (Fig. 5H-I). MiR-130a-3p decreased in human BC tissues, and miR-130a-3p in BC stem cells attenuated cellular proliferation, migration and invasion [21]. In the present study, Suv39H1 functioned as a direct target of miR-130a-3p, and HOTAIR enhanced Suv39H1 level by sponging miR-130a-3p in BC cells.
Suv39H1 plays a vital role in the proliferation and migration of prostate cancer cells [22]. Suv39H1 regulates the AKT/mTORC1 pathway in intestinal barrier homeostasis [23]. AKT induces the proliferation, invasion, and migration of cutaneous squamous cancer cells [24]. HOTAIR silencing enhanced the level of miR-130a-3p and reduced the expression levels of p-AKT, p-mTOR, PCNA, MMP9, and Bcl-2 in BC (C) SKBR3 cells with HOTAIR silencing that were transfected with Suv39H1 vector or empty vector were subjected to immunoblotting for Suv39H1, p-AKT, AKT, p-mTOR and mTOR expression.(D-F)HOTAIR silencing in SKBR3 cells significantly suppressed cell proliferation, migration and invasion. But Suv39H1 restoration promoted the proliferation, invasion, and migration of SKBR3 cells with HOTAIR knockdown. CCK8(D) and colony formation (E)assays were used to evaluate cell proliferation. Transwell invasion and migration assays(F) were performed to measure cell invasion and migration. (*p < 0.05 by Student's t-test, # p < 0.05 by ANOVA. n = three independent repeats.) cells (Figs. 5A and 6A). HOTAIR overexpression decreased the level of miR-130a-3p, increased the expression of Suv39H1, and activated the AKT/mTOR pathway in BC cells (Figs. 5B and 6B). MiR-130a-3p overexpression strikingly reduced the levels of Suv39H1 in BC cells (Fig. 5K). Suv39H1 restoration activated the AKT/mTOR pathway in SKBR3 cells with HOTAIR silencing (Fig. 6C).
HOTAIR reduced the level of miR-130a-3p, which inversely regulated the Suv39H1/AKT/mTOR pathway in BC cells. HOTAIR promoted the growth and metastasis of BC cells via the miR-130a-3p/Suv39H1 axis. Our results provide a better understanding of the interactions of HOTAIR and miR-103a-3p/Suv39H1 in BC and a potential prognostic biomarker and more effective clinical therapeutic target for BC.

Declaration of competing interest
The authors declare no competing financial interests.

Declaration of competing interest
The authors declare no conflict of interest.

Data availability
No data was used for the research described in the article.